The present invention provides novel methods and compositions for preparing and using electrochemical sensors to signal the presence of biological molecules.
Bacterial toxins are a primary cause of a variety of human diseases. For instance, some strains of Escherichia coli residing in the intestines of man and many other animals are capable of secreting various enterotoxigenic toxins. Indeed, these toxigenic strains of E. coli are generally considered as a cause of many diarrheal diseases (Scotland et al., Lancet i:90 [1980]). E. coli is the organism most commonly isolated in clinical microbiology laboratories, as it is usually present as normal flora in the intestines of humans and other animals. However, it is an important cause of intestinal, as well as extraintestinal infections. For example, in a 1984 survey of nosocomial infections in the United States, E. coli was associated with 30.7% of the urinary tract infections, 11.5% of the surgical wound infections, 6.4% of the lower respiratory tract infections, 10.5% of the primary bacteremia cases, 7.0% of the cutaneous infections, and 7.4% of the other infections (Farmer and Kelly, xe2x80x9cEnterobacteriaceae,xe2x80x9d in Manual of Clinical Microbiology, Balows et al.(eds), American Society for Microbiology, [1991], p. 365). Surveillance reports from England, Wales and Ireland for 1986 indicated that E. coli was responsible for 5,473 cases of bacteremia (including blood, bone marrow, spleen and heart specimens); of these, 568 were fatal. For spinal fluid specimens, there were 58 cases, with 10 fatalities (Farmer and Kelly, supra, at p. 366). There are no similar data for United States, as these are not reportable diseases in this country.
Studies in various countries have identified certain serotypes (based on both the O and H antigens) that are associated with the four major groups of E. coli recognized as enteric pathogens. Table 1 lists common serotypes included within these groups. The first group includes the classical enteropathogenic serotypes (xe2x80x9cEPECxe2x80x9d); the next group includes those that produce heat-labile or heat-stable enterotoxins (xe2x80x9cETECxe2x80x9d); the third group includes the enteroinvasive strains (xe2x80x9cEIECxe2x80x9d) that mimic Shigella strains in their ability to invade and multiply within intestinal epithelial cells; and the fourth group includes strains and serotypes that cause hemorrhagic colitis or produce Shiga-like toxins (or verotoxins) (xe2x80x9cVTECxe2x80x9d or xe2x80x9cEHECxe2x80x9d [enterohemmorrhagic E. coli]).
Detection of these toxins commonly involves the use of biological (i.e., animal) assays and immunoassays (for review, See, Jay (ed.), Modern Food Microbiology, Chapman and Hall, New York [1996]). Bioassays typically involve whole- or part-animal test, which are expensive and require a few days to complete. Imunological assays, on the other hand, couple antibody binding with optical signaling and amplification. The assay time can be reduced to one day or a few hours, depending on the type of method and toxin involved. Currently, various immunoassays are available for bacterial toxins include gel diffusion, reverse passive latex agglutination (RPLA) and enzyme-linked immunosorbent assay (ELISA) (See e.g., Pimbly and Patel, J. Appl. Microbiol. (Suppl.), 84:S98 [1998]). However, these techniques do not give results in a real-time detection fashion. In addition, RPLA can be affected by non-specific interference from materials which physically interfere the formation of a tight button (Mpamugo et al., J. Med. Microbiol., 43:442 [1995]), while ELISA is subject to enzymatic interference from samples containing peroxidase (Park et al., Appl. Environ. Microbiol., 60:677 [1994]). Poor antibody stability limits the use of these methods in field applications. An alternative approach for toxin detection involves the use of the polymerase chain reaction (PCR) to detect the bacterial gene(s) responsible for the production and release of toxins. While highly specific and sensitive, results from PCR may be inconclusive, as the gene may be present, but the toxin may be absent when the organisms are killed. Furthermore, inhibitors of DNA polymerases found in some samples may interfere with the detection of toxin.
Clearly, there is a need for fast, reliable, specific and sensitive methods for the detection of bacterial toxins for use in outbreak investigations, clinical diagnostics and quality monitoring in the food and feed industries. Recent progress in toxin detection methods has primarily focused on supramolecular assemblies (e.g., LB [Langmuir-Blodgett] monolayers and lipid bilayer membranes) coupled with specific cell surface receptors (See e.g., Charych et al., Chem. Biol., 3:113 [1996]). It has been shown that by engineering lipid membranes with desirable optical xe2x80x98reportingxe2x80x99 ability, colorimetric detection of cholera toxin (CT) can be achieved. The colorimetric sensor consists of self-assembly of the amphiphilic diacetylenic lipids and cell surface receptor GM1 in forms of LB monolayers and bilayer vesicles. Nonetheless, in spite of improvements in optical sensors, a still greater degree of sensitivity is needed in the art.
The present invention provides novel methods and compositions for preparing and using electrochemical sensors to signal the presence of biological molecules. It is not intended that the present invention be limited to any particular biological molecule. For example, it is contemplated that the present invention will find use in the detection and/or identification of microorganisms, prions, microbial toxins, antigens and/or antibodies, antigen and antibody complexes, and other suitable compounds or compositions of interest.
In some embodiments, the present invention is directed to novel biosensors for amperometric detection of E. coli heat-labile enterotoxin, in particular, enterotoxin LT. In preferred embodiments, the novel biosensor couples a redox supramolecular assembly with a sol-gel thin film electrode. In particularly preferred embodiments, the sensor utilizes an open platform to host biosensory elements, thereby allowing fast access of the target molecules to the redox vesicles for detection. Compared to other designs involving sol-gel encapsulation of enzymatic centers for biosensing, the diffusion process is greatly improved and molecular access is less restricted in the present invention. The measured apparent diffusion coefficients for lipid ferrocene are about 2-3 orders of magnitude higher than those for redox-doped polymer/gels. The response time and sensitivity can therefore be improved by the enhancement in mass transport.
The present invention also provides methods for using the amperometric biosensor. As described in the Examples below, the amperometric biosensor was demonstrated to have a detection limit of less that 3 parts per million, which is better than that obtained by colorimetric detection.
It is contemplated that the novel electrochemical sensors of the present invention will find use in the development of sensors for biological molecules such as proteins and toxins whose detection has previously relied upon other signaling mechanisms.
The present invention provides methods for measuring biomolecular recognition of at least one toxin by electrochemistry, comprising: providing liposomes having oxidation/reduction receptors selected from the group consisting of glycine-terminated diacetylene lipid, acetylferrocenic diacetylene lipid, and a glycosphingolipid known to be a receptor for at least one toxin; adding a sample suspected of containing at least one toxin to the liposomes; and measuring the anodic current of ferrocene in order to determine the biomolecular recognition of at least one toxin by the liposomes. In some embodiments, the sample is known to contain at least one toxin or other analyte(s), while in others it is unknown whether the sample contains any toxins or analytes at all. In alternative embodiments, at least one of the toxins comprises an enterotoxin. In other embodiments, only enterotoxin is present in the sample. In some preferred embodiments, the enterotoxin is E. coli enterotoxin, while in some particularly preferred embodiments, the receptor is a receptor for 84-kDa E. coli enterotoxin. In further embodiments, the measuring is conducted by voltammetric determination.
The present invention also provides methods measuring biomolecular recognition of a toxin by electrochemistry, comprising: providing liposomes having oxidation/reduction receptors selected from the group consisting of glycine-terminated diacetylene lipid, acetylferrocenic diacetylene lipid, and a glycosphingolipid known to be a receptor of 84-kDa E. coli enterotoxin, and a sample suspected of containing E. coli enterotoxin; adding the sample to the liposomes; and measuring the anodic current of ferrocene in order to determine the biomolecular recognition of E. coli enterotoxin by the liposomes. In some preferred embodiments, the mixture ratio of glycine-terminated diacetylene lipid, acetylferrocenic diacetylene lipid, and a glycosphingolipid known to be a receptor of 84-kDa E. coli enterotoxin is approximately 4:1:0.25 respectively. In some embodiments, the sample is known to contain E. coli enterotoxin , while in others it is unknown whether the sample contains E. coli enterotoxin. In still further embodiments, the sample may contain E. coli enterotoxin as well as additional toxins or other analytes. In still other embodiments, the measuring is conducted by voltammetric determination.
The present invention further provides methods for measuring biomolecular recognition of an analyte by electrochemistry, comprising: providing liposomes having oxidation/reduction receptors selected from the group consisting of: glycine-terminated diacetylene lipid, acetylferrocenic diacetylene lipid, and a glycosphingolipid known to be a receptor of an analyte, a sample suspected of containing at least one analyte; adding the sample to said liposomes, and measuring the anodic current of ferrocene to determine the biomolecular recognition of the analyte by said liposomes. In some embodiments, the analyte is selected from the group consisting of microorganisms, drugs, receptor ligands, antigens, allergens, ions, hormones, blood components, disease indicators, cell components, antibodies, lectins, enzymes, organic solvents, volatile organic compounds, pollutants, and genetic material. In some embodiments, the sample is known to contain an analyte, while in others it is unknown whether the sample contains an analyte. In still further embodiments, the sample may more than one analyte (e.g., an analyte of particular interest, as well as other analytes, which may or may not be of interest). In yet other embodiments, the microorganism is selected from the group consisting of viruses, bacteria, parasites, fungi, and prions. In some preferred embodiments, the microorganism is a pathogen. In some alternative embodiments involving viruses, the virus is selected from the group consisting of influenza, rubella, varicella-zoster, hepatitis A, hepatitis B, herpes simplex, polio, small pox, human immunodeficiency virus, vaccinia, rabies, Epstein Barr, reoviruses, and rhinoviruses. However, it is not intended that the present invention be limited to any particular virus or analyte.